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The aim of the present study was to investigate the effect of melatonin on the isolated rat anococcygeus muscle (RAM). The RAM was isolated as described by Gillespie 1971. The muscles were mounted in an organ bath containing modified Krebs-Henseleit solution at 37°C and, changes in the tension due to the precontractile agents and melatonin were measured by isometric force displacement transducers. Although cumulative addition of melatonin (10 -10-10-3 M) produced no effect on the basal tone of the anococcygeus muscle, it caused an inhibitory action on the muscle pre-contracted by KCl (70 mM) and phenylephrine (1.7×10-6 M). In the sodium fluoride (NaF) (3×10-3 M)-precontracted muscles, melatonin (10-5-10-3 M)-induced maximum relaxation was found higher than that obtained in phenylephrine or KCl-precontracted RAM. Incubation with phorbol myristate acetate (PMA (10-10-10-4 M) an activator of protein kinase C) produced no effect on the resting or precontracted muscle. In conclusion, the inhibitory effect of melatonin might be related to the G-protein mediated pathways, but not to protein kinase C.